Int. J. Adv. Multidiscip. Res. (2017).

4(2): 27-32

International Journal of Advanced Multidisciplinary Research

ISSN: 2393-8870
www.ijarm.com
DOI: 10.22192/ijamr Volume 4, Issue 2 -2017
Research Article DOI: http://dx.doi.org/10.22192/ijamr.2017.04.02.004

Caulogenic response of in vitro raised nodal explants of

Orthosiphon stamineus to selected auxins

R. Elangomathavan*, P. Kalaivanan, S. Hariharan and S. Nancy Beaulah

Department of Biotechnology, PRIST University, Thanjavur, Tamilnadu-613403, India
*Corresponding author:
Dr. R. Elangomathavan, Assistant Professor, Department of Biotechnology, PRIST University, Thanjavur,
Tamilnadu, India. E-mail: relangomathavan@gmail.com; Telephone No: +919884289207

Abstract

Keywords
Auxin,
nodal explants,
micropropagation,
shoot elongation and
Orthosiphon stamineus
The effect of auxins on in vitro raised nodal explants of Orthosiphon stamineus cultured in
MS medium for the study of shoot initiation and elongation. Shoot induction and callus
induction development depended on the type of explants (node, internode andleaf) while
exposed to MS medium addition with different concentration of 2, 4-D, NAA and IBA plant
growth hormones. Nodal explants showed shoot induction at 0.5 mg/l, 1.0 mg/l, and 2.0
mg/l concentration of 2, 4-D, IBA, and NAA. Among these hormones 2, 4-D showed higher
(92.8%) shooting response with 18.2 numbers of nodes per shoot and 16.6 cm shoot height
followed by NAA and IBA at 0.5 - 3.0 mg/l concentrations. Similarly other explants such as
internode and leaf were inoculated in MS medium with the same concentrations of
hormones for the identification of morphological changes. After 25 days, leaf and internode
explants induced callus. Similarly maximum number of root induction was observed in ¼
MS medium with 1.0 mg/l concentrations of IBA. The present study claims that the
effectiveness of hormone concentration and explants efficiency for the mass propagation of
the O. stamineus plant.

Introduction
Auxins play an important role in stem initiation and
elongation which involves in different features of
growth and developments in higher plants. The auxins 2,
4-D are strong promoters of callus induction and growth
of cell suspensions. There are only a few examples of
the shoot and root induction by phenoxy auxins in tree
tissue cultures (Zaerr and Mapes, 1982). For shoot
induction generally requires the combination of auxin
and cytokinin. However, auxin should be used carefully
since too much auxin favors callus growth. Moreover,
for shoot initiation in some explants, the production of
endogenous auxin is sufficient for induction of shoots in
larix decidue (Bondga and Von-Aderkas, 1992). The
number of shoots induced on MS medium (Murashige
and Skoog, 1962). The auxin signal is predictable by
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plant cells and rapidly converted to a wide variety of
responses in the growth and development of plant
organs. These comprise alters in the direction of growth,
shoot and root branching, and vascular differentiation
(Leyser, 2001).
The plant cell division and growth of tissue, cells
cultured in vitro require an external source of auxin
(Petrasek et al., 2002). The proportion of external to
internal auxin concentrations is essential for regulation
of the different stages of the standard growth cycle. The
type of auxin used in the medium influences culture
morphology (Hofmann, et al., 2004). Most of the plants
naturally contain cytokinins such as 6-furfuryl-
aminopurine, Ribosyl zeatin, Zeatin Isopentenyl adenine
 Int. J. Adv. Multidiscip. Res. (2017). 4(2): 27-32
and Dihydro zeatin. These endogenous cytokinins
interact with exogenous auxin 2, 4-D may lead to
shoot induction. Auxin pulse produced a clear effect
improving regeneration (Pascual and Marin et al.,
2005). Indeed, auxins obviously involved in
morphological changes since it regulates plant cell
division, elongation, and differentiation (Chen, 2001).
The effect of a liquid 2, 4-dichlorophenoxyacetic acid
(2, 4-D) influence on the adventitious regeneration of
both shoots and roots in the regeneration medium
containing 2, 4-D concentration (Pascual and Marin et
al., 2005). Numerous alterations occur in plant
morphology by lighting conditions and altering
hormone composition in the regeneration medium
(Gentile et al., 2002).
Orthosiphon stamineus is a medicinal plant belongs to
Lamiaceae which is distributed mainly in South East
Asian countries. This plant was reported to have
secondary metabolites with biological activity
properties; therefore, it has a great potential value for
the development of this plant through in vitro
propagation. This may help to develop an alternative
documentary repository protocol for this plant. In the
past, few micropropagation protocols have been
developed using MS medium with an auxin-cytokinin
combination of different concentrations for this plant;
alternatively, this protocol may influence the ability to
develop whole plants. The aim of this study was to
develop a new protocol for the development of whole
plantlets with more number of nodes in a single shoot
using MS medium with different concentrations of
auxins such as 2, 4-D, NAA and IBA (0.5 – 3.0 mg/l)
on nodal explants of O. stamineus.
Materials and Methods
In vitro raised O. stamineus plantlets were excised into
nodal explants by using sterile scalpel blade and
forceps, and then inoculated aseptically on MS
medium with 3% sucrose. The devoid of growth
regulators were served as control and MS medium
with (0.1 - 3.0 mg/l of 2, 4-D, NAA, and IBA
separately) different concentration of auxins were
served as a hormone-treated medium. The pH was
adjusted to 5.7 and medium was solidified by adding
0.6% agar then autoclaved for 20 minutes at 121ºC. In
vitro cultured nodal explants were used for all the
treatments and five replicates were used. All the
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cultures were grown under a 16 h photoperiod by cool
white fluorescent lamps with 48μmol.m-2s-1 photon
flux density and 8 h dark condition at 25ºC. The
maximum number of nodes, height of shootlets and
rooting were recorded after 25 days of incubation
period.
Data analysis
Shoot induction was recorded based on the percentage
of nodal explants development on MS medium
supplemented with different auxin concentration in the
time period of 25 days of inoculation. A randomized
statistical design was performed using SPSS 13 (SPSS
Inc., Chicago, IL, USA) and Excel 2007 (Microsoft,
Redmond, WA, USA) software. single factor ANOVA
data was performed and analyzed the statistical
significant and least significant differences (LSD) test
was developed to compare means at p<0.05 level.
Results and Discussion
Caulogenic response
The in vitro raised nodal explants were inoculated on
MS medium with a different concentration of each
auxins (2,4-D, NAA and IBA) (Table 1). All the three
auxins induced the nodal explant to generate single
shoots rather than developing clump of multiple
shootlets which is a normal response of cytokinin.
However the nodal explants gave single shoot the
shoot height is significant and remarkable outcome in
this experiment. Of the various auxins used maximum
shooting response (92.8%) and maximum shoot height
(16.6 cm) with 18.6 mean number of nodes per shoot
was obtained from the explants cultured on MS + 0.5
mg/l 2, 4-D (Table 1; Fig 1). It is obvious that only at
low concentration of auxin regime was suitable for the
production of elongated shoots with more number of
nodes. Increase in the concentration of auxin
reciprocally decrease the production of shootlet with
remarkable height and also very few number of node
per shootlet proportionally. Hormone free medium
containing nodal explants showed less (5%) shoot
induction response and at high concentration of auxin
(3.0 mg/l) did not induce any shoot development;
instead of that callus induction was developed along
with small adventitious rootlets.
 Int. J. Adv. Multidiscip. Res. (2017). 4(2): 27-32
Table 1: Impact of auxins on caulogenic response of nodal explants cultured in MS
medium and data were recorded after 25 days of inoculation.

Mean Shoot
MS + Plant growth
Shooting number of height
plant regulators
response node per (cm) ±
growth concentration
(%) regenerated S.D*
regulators (mg/l)
shoot ± S.D*
Control 0 5.0 ± 0.3 2.8 ± 0.5f 3.5 ± 0.5
0.5 93.0 ± 1.2 18.6 ± 0.6a 16.6 ± 0.4
1.0 80.0 ± 1.6 12.2 ± 0.5b 14.7 ± 0.4
2, 4-D
2.0 21.0 ± 0.7 6.8 ± 0.4d 4.8 ± 0.3
3.0 - - -
0 4.8 ± 0.2 2.6 ± 0.5f 3.5 ± 0.2
0.5 60.0 ± 0.9 13.0 ± 0.7b 4.9 ± 0.1
d
NAA 1.0 35.0 ± 0.4 7.8 ± 0.4 3.5 ± 0.2
2.0 10 .0 ± 0.3 4.6 ± 0.5e 3.0 ± 0.7
3.0 - - -
0 4.6 ± 0.5 2.6 ± 0.5f 3.2 ± 0.4
0.5 32.0 ± 0.5 10.4 ± 0.8c 3.3 ± 0.2
IBA 1.0 20.0 ± 0.4 7.2 ± 0.6d 2.3 ± 0.2
2.0 5.0 ± 0.3 4.2 ± 0.5e 1.9 ± 0.1
3.0 - - -
*Values are mean ± Standard Deviation (n = 5), dissimilar letters indicated significant differences
between means within treatments at p<0.05 level based on LSD mean separation.

Other explants such as internodes and leaf did not
show any shoot induction however callus induction
was observed along with few adventitious rootlets. In
our studies, shoot induction was observed in the nodal
explants under the lower concentration of 2, 4-D.
Similarly Popielarska et al., (2006) reported in
Brassica napus. Cv. and Mello et al., (2001) in
Curcuma zedoaria. The endogenous auxin produced
on the apical shoot tip is used to be transported
downward to the basal part of plants like stem and root
region (Terasaka et al., 2005). At low concentration of
2, 4-D helps in shoot elongation and also produce
more number of nodes per new single regenerated
shootlet. Those in vitro raised nodes may be explored
for the mass propagation of the plant by undergoing
many sub or re-culture methods with short interval
time.
The morphological changes on cultured tissue may be
linked to the composition of the culture medium. It is
recognized that culture conditions, as well as the plant
genotype, have a high impact on shoot induction
frequency (Hu et al., 1999). Shoot development was
observed on hypocotyls cultured on 2, 4-D-
supplemented media (Popielarska et al., 2006); (Khan
et al., 2002). Our observations pointed out that the
response of nodal explants in hormone-free medium
and hormone (2, 4-D, NAA and IBA auxins) treated
medium showed enormous differences in
organogenesis for this plant. However, some shoots
emerged in the nodal bud and become necrosis after
few days of inoculation. Similar results were reported
in bean (Angelini and Allavena, 1989). The decline of
2, 4-D concentration may lead to the shoot induction
because novel genes are switch on for plant
regeneration. This harvest may only be synthesized
when exogenous auxin (2, 4-D) is greatly reduced
from the medium (Michalczuk et al., 1992a);
(Zimmerman 1993). In earlier studies, BAP hormone
concentrations were reduced from 1.0 to 0.1 mg/l to
avoid the poor effects of shoot induction by long term
exposure to high concentrations (Vieitez et al., 1985;
San-Jose et al., 1988; Chalupa, 1988). Similarly at low
concentration of 2, 4-D hormone favors shoot
elongation within short duration of time period.

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 Int. J. Adv. Multidiscip. Res. (2017). 4(2): 27-32

Fig: 1. (A) Shoot regeneration in nodal explants and callus induction in internode & leaf explants from O. stamineus
cultured in MS medium (control). (B) Shoot regeneration at 0.5 mg/l 2, 4-D (C) Shoot elongation at 0.5 mg/l 2, 4-D
(D) Shoot elongation at 1.0mg/l 2, 4-D. (E) hairy root induction at 1mg/l IBA.

Root induction induction was developed in the IBA treatment for

The regenerated shoots were transferred to ¼MS
medium with auxins at 1.0 mg/l IBA induced 19.4
numbers of rootlets with 94% of rooting response. The
role of explants and auxin hormone concentrations
take part in an importance in the MS medium for the
effectiveness of root induction or inhibition under
suitable conditions (Eduardo, 1998). High root
Ginseng (Choi et al., 1994). Thus, it was suggested
that the exogenous supply of IBA induces more
number of rootlet induction for this plant specious.
Although at high concentration of auxins IBA, IAA
and NAA (>3 mg/l) lead to decline of rooting which
may be due to the herbicidal activity of auxins at high
concentration (Evan et al., 2003).

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 Int. J. Adv. Multidiscip. Res. (2017). 4(2): 27-32
Table 2: Shows the rooting response of the in vitro raised shootlet of O. stamineus cultured on ¼ MS medium.

Percentage of Mean number of

¼ MS + Conc. Root length
rootlet rootlets/ shoot ±
PGRs (mg/l) (cm) ± S.D*
induction S.D*
Control 0 21.0 ± 2.2 2.8 ± 0.2g 1.6 ± 0.6
0.5 68.2 ± 2.4 9.0 ± 0.7de 2.8 ± 0.4
1.0 84.0 ± 2.2 16.8 ± 0.8b 3.8 ± 0.4
IAA
2.0 69.4 ± 1.9 8.0 ± 0.7e 4.6 ± 0.5
3.0 45.2 ± 1.4 5.2 ± 0.4f 3.2 ± 0.4
g
IBA 0 20.4 ± 1.9 2.2 ± 0.4 1.8 ± 0.4
0.5 71.0 ± 3.1 11.4 ± 0.9d 4.6 ± 0.5
1.0 94.2 ± 2.4 19.4 ± 0.5a 11.4 ± 0.9
2.0 70.4 ± 1.5 15.2 ± 0.8b 8.4 ± 0.5
d
3.0 51.2 ± 2.2 10.4 ± 0.6 5.0 ± 0.7
0 20.2 ± 1.4 2.6 ± 0.5g 2.2 ± 0.4
0.5 66.0 ± 2.5 8.2 ± 1.1e 3.2 ± 0.4
NAA 1.0 76.4 ± 3.2 13.6 ± 0.5c 5.0 ± 0.7
2.0 50.2 ± 1.8 6.4 ± 0.5f 5.2 ± 0.4
f
3.0 35.8 ± 2.3 5.0 ± 0.7 3.6 ± 0.5
*Values are mean ± Standard Deviation (n = 10), dissimilar letters indicated significant differences between means
within treatments at p<0.05 level based on LSD mean separation.

In conclusion, we found that 2, 4-D involves more
effective to develop shoot induction and elongation at
an optimized concentration in O. stamineus. These
studies have shown that the threshold level of 2, 4-D
for shoot induction seems to be 0.5-1.0 mg/l. Within
this range, the explants cultured in lower concentration
of 2, 4-D in MS medium to develop more number of
nodes in a single shootlet which favours subculture to
enhance the rate of mass propagation within short
duration of time.
Acknowledgments
The authors express their gratitude to the management
of PRIST University for having given permission and
facility to carry out the research work.
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DOI:10.22192/ijamr.2017.04.02.004

How to cite this article:

R. Elangomathavan, P. Kalaivanan, S. Hariharan and S. Nancy Beaulah. (2017). Caulogenic response of in
vitro raised nodal explants of Orthosiphon stamineus to selected auxins. Int. J. Adv. Multidiscip. Res. 4(2):
27-32.
DOI: http://dx.doi.org/10.22192/ijamr.2017.04.02.004

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